| There are three sections in this dissertation: 1) quantification of gene expression in chicken embryo fibroblasts (CEF) and virus load in bursal Fabricius (BF) infected with infectious bursal disease virus (IBDV), the recombination of infectious laryngotracheitis virus (ILTV) and pathotyping of Newcastle disease virus (NDV).Section one:IBDV can cause disease in chickens characterized by immunosuppression and high mortality. Quantitative real time reverse transcription polymerase chain reaction (RT-PCR) has been previously applied as a sensitive, reproducible and rapid method for detection and quantification of nucleic acid. In this study, quantitative real time reverse transcription polymerase chain reaction (RT-PCR) was applied to determine one constant expression gene which could be used as internal control to quantify gene expression and virus load, and then to study virus replication and gene expression of CEF cells infected with IBDV, as well as to quantify IBDV in bursa of Fabricius and feces, which taken from chicken experimentally infected with IBDV.Firstly, in order to select an internal control for quantitative real time RT-PCR, the expression of twelve genes of chicken embryo fibroblasts (CEF) including genes encoding P-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP), B-2-microglobulin, ovotransferrin, Ubiquitin C (UBC), Dead box protein (DDX1), IL-13Ra G6PDH and PRKG were investigated during a 7-day period after inoculation with IBDV by use of quantitative real time RT-PCR with SYBR green dye. Of the twelve genes, P-actin, 28S rRNA, 18S rRNA and GAPDH were constantly expressed during the infection. The p-actin gene was shown to be of moderate expression and did not show significant differences in normalized and non-normalized assays. Therefore this gene is a candidate as internal control for studying gene expression in CEF infected with IBDV as well as IBDV load in CEF during infection.The P-actin was used as internal control in quantitative real time RT-PCR with SYBR green dye to quantify expression of genes of CEF cells during a 7-day period after inoculation with IBDV, these genes include TNF (Tumor necrosis factor) -related apoptosis inducing ligand-like protein (TRAIL-like), apoptosis inhibitor l,Stat3, macrophage inflammatory protein-3alpha (MIP-3alpha), IL-6, IL-8, interferon regulatory factor 1 (IRF 1), gibbon ape leukemia virus receptor 1 (GLVR1). TVB receptor, bovine leukemia virus receptor p38 (BLVRcp38), interferon alpha/beta receptor 1 (IFNAR1), interferon alpha/beta receptor 2 (IFNAR2). IFN 1/2 promoter, 2',5'-oligo adenylate synthetase A (2,5-A), MHC class I and class II, Nuclear factor kappa B p65 (NFkB p65) and alpha-2,3-sialyltransferase (ST3GAL-VI). Of these genes, Stat 3, MIP-3 a , IL-8, IFN 1-2 promoter, 2, 5-adenylate synthetase A, MHC class 1, MHC class 2, IRF-1 and TVB receptor were up-regulated morethan 10 times; apoptosis inhibitor 1, IFNAR1, ALVR1, a 2.3-alpha-2,3-sialyltransferase, IL-6 were up-regulated less than 10 time; NF k B p65, BLRcp38, IFNAR2 were expressed constantly; TRAIL-like was down-regulated by infection of IBDV. All the up-regulated genes showed dose of infection dependent, those infected with high titer of virus were obviously up-regulated at early stage other than those of low titer of infection. The possibility of these genes in antiviral reaction in host and the pathway of host response to virus infection were being discussed.Quantitative real time RT-PCR with SYBR green dye were applied to quantify and detect the virus load in bursa of Fabricius and feces, which were taken from chicken infected with vaccine strain D78, very virulence strain IBDV (wIBDV, Denmark), Classical strain F52/70, separately. All the strains could be detected in bursa of Fabricius at day 1 post infection (p.i.) and in feces (except D78) at day 2 p.i. The wIBDV and D52/70 showed high amount relative to B-actin in bursa of Fabricius at day 2 and 3 post infection and in feces at day 3, the D... |
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